The alpha-crystallins, which comprise a large fraction of the soluble protein in the vertebrate lens, where they are believed to function solely as structural proteins, are the first crystallins to be expressed in the developing mouse lens and are a relatively small family of crystallins encoded by only two genes, the alphaA- and alphaB-crystallin genes. The alpha-crystallins exhibit molecular chaperon activity and, at least in the case of alphaB-crystallin, have been shown to be expressed in a variety of nonlenticular tissues, where their function is unknown. Toward understanding the role of the alpha-crystallins in lens and nonlens tissues, we are attempting functional deletion of alpha- crystallins by disrupting the alpha-crystallin genes in mice. We are employing the technique of homologous recombination in pluripotent mouse embryonic stem cells, followed by the generation of chimeric mice containing the altered stem cells. We have isolated and mapped 15-kb clones containing the alphaA- and alphaB-crystallin gene loci from a mouse 129SV library (the same strain as most of the embryonic stem cells lines currently in use). Construction of the alphaA-crystallin "knockout vector" is near completion. In collaboration with Dr. Nicholas Ambulos (University of Maryland Medical School), we have nearly completed double- stranded sequencing of the mouse alphaA-crystallin gene (5 kb) and single-stranded sequencing of an additional 4 kb of 5'-flanking sequence. We currently are generating constructs to look for enhancer regions in the alphaA-crystallin introns and in the 5'- and 3'-flanking regions.